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Novel chimpanzee adenovirus-vectored respiratory mucosal tuberculosis : overcoming local anti-human adenovirus immunity for potent TB protection

M Jeyanathan1,3, N Thanthrige-Don1,3, S Afkhami1,3, R Lai1, D Damjanovic1, A Zganiacz1, X Feng1, X-D Yao1, KL Rosenthal1, M Fe Medina1, J Gauldie1, HC Ertl2 and Z Xing1

Pulmonary tuberculosis (TB) remains to be a major global health problem despite many decades of parenteral use of Bacillus Calmette–Gue´ rin (BCG) vaccine. Developing safe and effective respiratory mucosal TB represents a unique challenge. Over the past decade or so, the human serotype 5 adenovirus (AdHu5)-based TB vaccine has emerged as one of the most promising candidates based on a plethora of preclinical and early clinical studies. However, anti-AdHu5 immunity widely present in the lung of humans poses a serious gap and limitation to its real-world applications. In this study we have developed a novel chimpanzee adenovirus 68 (AdCh68)-vectored TB vaccine amenable to the respiratory route of . We have evaluated AdCh68-based TB vaccine for its safety, T-cell immunogenicity, and protective efficacy in relevant animal models of human pulmonary TB with or without parenteral BCG priming. We have also compared AdCh68-based TB vaccine with its AdHu5 counterpart in both naive animals and those with preexisting anti-AdHu5 immunity in the lung. We provide compelling evidence that AdCh68-based TB vaccine is not only safe when delivered to the respiratory tract but, importantly, is also superior to its AdHu5 counterpart in induction of T-cellresponses and immune protection, and limiting lung immunopathology in the presence of preexisting anti-AdHu5 immunity in the lung. Our findings thus suggest AdCh68-based TB vaccine to be an ideal candidate for respiratory mucosal , endorsing its further clinical development in humans.

INTRODUCTION after birth in most countries. Although BCG effectively protects Pulmonary tuberculosis (TB) caused by Mycobacterium against disseminated childhood TB, it has failed to effectively tuberculosis (M.tb) has haunted mankind for thousands of control adolescent and adult pulmonary TB.3,5 Thus, there is an years and still remains a top infectious killer.1–4 TB causes B1.4 urgent need to develop novel TB vaccines that can be used for million deaths and 9 million new cases each year. An estimated effective boost vaccination following parenteral BCG priming one-third of the world population is latently infected by M.tb in humans.6 and 5–10% of these people develop active TB some time in their One of the immune evasion strategies that M.tb utilizes is to lives. Despite availability of antibiotics, increased multidrug- significantly slow down the appearance of T-cell immunity in resistant or extensively drug-resistant TB cases continue to pose the lung.7 Such delay or ‘‘immunological gap’’ allows M.tb a a global threat. Bacillus Calmette–Gue´rin (BCG), which has foothold in the lung before immunity is established,7–10 and this been used in humans for many decades, remains the only is likely one of the reasons for the ineffectiveness of parenteral licensed anti-TB vaccine. BCG is given once via the skin shortly BCG vaccination in humans.6,7 Therefore, it is believed that

1McMaster Immunology Research Centre, Department of Pathology and Molecular Medicine and Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada and 2Department of Immunology, The Wistar Institute, Philadelphia, Pennsylvania, USA. Correspondence: Z Xing ([email protected]) 3The first three authors contributed equally to this work. Received 25 September 2014; accepted 20 March 2015; published online 15 April 2015. doi:10.1038/mi.2015.29

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effective boost vaccination strategies ought to fill such a gap in hepatitis C virus.33,35–39 However, up to date, AdCh vector T-cell immunity in the lung.6,7,11,12 Recent preclinical research technology has not been translated to developing TB vaccines. from us and others has identified the respiratory mucosal route Furthermore, its potential for respiratory mucosal application of vaccination to be the most effective way to equip the lung, and the effect of pulmonary anti-AdHu5 immunity on its particularly the surface of the respiratory mucosa (the airway potency are still poorly understood. luminal compartment), with protective T-cell immunity.6,11–14 In this study, for the first time we have applied the new Mucosal vaccination does so via activating mucosal-specialized technology for developing a novel replication-defective AdCh- dendritic cells and upregulating mucosa-homing molecules based TB vaccine for respiratory mucosal application. By using on T cells.10,15,16 Nevertheless, almost all of the TB vaccine preclinical models of human pulmonary TB with or without candidates currently in the TB vaccine pipeline are still being preexisting anti-AdHu5 immunity, we provide compelling developed as parenteral vaccines for human application.4 In evidence that respiratory-mucosally delivered AdCh-based TB this regard, the immune adjuvant used for parenteral delivery of vaccine activates high levels of T-cell responses, provides robust protein-based TB vaccines is unsafe for respiratory mucosal protection, and limits lung pathology at a capacity com- applications.17,18 Replicating mycobacterial-based TB vaccines parable to or even better than its AdHu5 counterpart. are known to cause granulomatous inflammation and cannot Importantly, contrary to AdHu5Ag85A, respiratory mucosal be easily cleared from the lung, thus deemed unsuitable for AdCh68Ag85A-induced protective T-cell immunity is mini- respiratory mucosal vaccination. Recent report on the inability mally affected by anti-AdHu5 immunity preexisting locally in of intradermally delivered MVA (modified vaccinia Ankara)- the lung. Our study supports the AdCh-based TB vaccine to be based TB vaccine to enhance protective immunity in a major an ideal candidate for respiratory mucosal immunization in milestone phase 2b trial19 speaks further to the urgency and the humans and thus warrants its further clinical development. importance of the effort in developing different vaccination strategies such as mucosal vaccination against pulmonary TB. RESULTS However, developing both safe and effective respiratory Molecular construction and characterization of a mucosal-deliverable vaccines represents a unique challenge and chimpanzee adenovirus vector AdCh68-based TB vaccine has recently been identified, at the National Institutes of Health A replication-deficient chimpanzee type 68 adenovirus was (NIH)/Aeras Aerosol TB Vaccine Workshop, to be a new 6,13–15,20 constructed to encode a secreted form of Ag85A, a highly priority. Recombinant, replication-deficient virus-vec- immunogenic secreted antigen of M.tb (AdCh68Ag85A), by tored TB vaccine platforms represent the most attractive 40 12,21–23 using a recently described direct cloning technology (Figure 1a). approach for respiratory mucosal vaccination. Based on Expression of Ag85A is under control of a murine its unrivaled potency, the group C human serotype 5 adenoviral cytomegalovirus promoter and its secretion by mammalian vector system (AdHu5) has been widely used for developing 21,22,24–26 cells is promoted by a human tissue plasminogen signal peptide vaccines against infectious diseases and cancer. sequence (tissue plasminogen activator). All Ag85A genetic Indeed, an AdHu5-based TB vaccine developed by us was expression elements in AdCh68Ag85A are identical to those in robustly protective, particularly when given via the respiratory 41 6,12,21 its human type 5 adenovirus-based TB vaccine AdHu5Ag85A. tract, in a number of animal models. This vaccine has also AdCh68Ag85A was packaged and propagated in 293 cells and recently been evaluated following intramuscular administra- 27 purified under the Good Laboratory Practice conditions. Using tion in a phase 1 clinical study. However, a major gap and a monoclonal antibody specific for Ag85A, the production and limitation to the application of AdHu5-based vaccine to secretion of Ag85A protein were characterized in lung epithelial humans is the pervasive preexisting anti-AdHu5 immunity A549 cells by western immunoblotting. The expected size of resulting from respiratory exposure to AdHu5 virus that not B30 kDa Ag85A protein was detected in both the culture only compromises the potency of the vaccine but may also pose supernatant and cellular lysate from A549 cells infected with a safety concern when given to HIV high-risk popula- 22,24,25,28–32 AdCh68Ag85A but not from those infected with empty vector tions. Although human adenoviruses of rare sero- AdCh68 (Figure 1b). The levels of Ag85A antigen production types such as AdHu35 may circumvent this issue, such vector is by AdCh68Ag85A were found to be comparable to those from poorly immunogenic because of its strong type 1 interferon 33–35 the cells infected with the same dose of AdHu5Ag85A (IFN)-inducing ability. (Figure 1b). These observations form the basis to compare Recently, replication-defective chimpanzee-derived adeno- AdCh68Ag85A with AdHu5Ag85A for their safety, viruses (AdCh) have become an attractive alternative to human immunogenicity, and protective efficacy in vivo. adenoviral counterparts.33,35 Neutralizing antibodies against 29,33–35 AdCh vectors are rarely found in humans. Furthermore, Respiratory mucosal with AdCh68Ag85A elicits as most AdCh viruses utilize the same cell entry receptors as desired innate immune activation and minimal tissue AdHu5, its immunogenicity is comparable to that by AdHu5. inflammation in the lung The large-scale production is also readily achievable as the same To begin evaluating the suitability of AdCh68Ag85A for viral packaging cell line for AdHu5 can be used for amplifying respiratory mucosal vaccination, we first assessed to which AdCh vectors. Thus, AdCh technology has recently been degree the vector induced an inflammatory reaction in the lung. exploited to develop vaccines for malaria, HIV, Ebola, and To this end, mice were inoculated intranasally (i.n.) with

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innate immune responses in the lung, we analyzed the major cellular components in bronchoalveolar lavage fluid (BAL). The majority of innate immune cells in the BAL of AdCh68Ag85A lungs were alveolar macrophages at days 1 and 3(Figure 2b). A degree of transient neutrophilic responses was observed only at day 1 but hardly at day 3 (Figure 2b). We also examined the responses of innate immune cytokines tumor necrosis factor-a and IFNb in the BAL. Tumor necrosis factor-a is a type 1 immune cytokine,7 whereas IFNb is a type I interferon usually induced during viral and has been shown to suppress anti-TB T helper type 1 cell immunity.42–44 Although intranasal inoculation of AdCh68Ag85A triggered a level of tumor necrosis factor-a production, the level of IFNb was very low at the both time points (Figure 2c,d), in agreement with our previous finding.42 Upon comparison we found that the proinflammatory responses induced by AdCh68Ag85 were largely comparable to its human AdHu5Ag85A counterpart in histopathology (Figure 2a), inflammatory cellular responses (Figure 2b), and innate immune cytokine responses (Figure 2c,d). The above data together suggest that although AdCh68Ag85A possesses desired immune adjuvant properties, it is safe for respiratory mucosal administration. Furthermore, like its AdHu5 counter- part, AdCh68Ag85A triggers a negligible level of undesired type 1 IFN responses.

Respiratory mucosal AdCh68Ag85A immunization induces robust T-cell responses in the lung We next examined the T-cell immunogenicity of AdCh68Ag85A following respiratory mucosal vaccination. The mice were vaccinated i.n. once with AdCh68Ag85A as described above. For comparison, some mice were immunized i.n. with an equal dose of AdHu5Ag85A. T-cell responses were analyzed in the BAL and lung tissue at 2 and 4 weeks after vaccination (Figure 3a) by Ag85A tetramer (tet þ ) and intracellular IFNg (IFNg þ ) immunostaining. AdCh68Ag85A vaccination induced high levels of Ag85A tetramer-positive CD8 T cells Figure 1 Molecular construction and characterization of AdCh68Ag85A. (a) An Ag85A genetic cassette was cloned into the (tet þ CD8 þ ) in the airway lumen (BAL) and lung interstitium pShuttle plasmid and was then excised with the indicated enzymes. The (Lung) at 2 weeks (Figure 3b). By 4 weeks, the magnitude of I-Ceu1 and PI-Sce1-excised insert was subcloned into the I-Ceu1 and tet þ CD8 T cells contracted significantly. AdCh68Ag85A PI-Sce1 site of pAdC68 genomic clone to generate recombinant AdCh68Ag85A virus. (b) Characterization of Ag85A protein expression vaccination induced similarly increased responses of IFNg- levels by AdCh68Ag85A-infected A549 cells was performed in culture producing CD8 T cells in both the BAL and lung upon stimu- supernatant and cellular lysate by using western blots and compared lation with an immunodominant Ag85A peptide (Figure 3c). with the cells infected with AdHu5Ag85A. The empty viral vectors Addl70-3 and AdC68 were used as negative controls for AdHu5Ag85A and AdCh68Ag85A vaccination also increased CD4 T-cell res- AdCh68Ag85A, respectively. Recombinant Ag85A protein was used as a ponses but at much lower levels than CD8 T-cell responses. In positive control ( þ ve). MCMV, murine cytomegalovirus promoter, tPA, comparison, although AdHu5Ag85A vaccination also induced tissue plasminogen activator. potent CD8 T-cell responses in the BAL and lung, the overall levels of T-cell responses at 2 weeks in the BAL were signifi- cantly lower than AdCh68Ag85A. Similar to AdCh68Ag85A, T-cell responses contracted significantly at 4 weeks after AdCh68Ag85A. As a comparison, a group of mice were AdHu5Ag85A vaccination (Figure 3b,c). inoculated with an equal dose of AdHu5Ag85A. By histo- To further evaluate the functionality of respiratory mucosal pathological examination at days 1 and 3 after AdCh68Ag85A, CD8 T cells activated by intranasal AdCh68Ag85A vaccination, only mild inflammatory infiltrates were seen in the peripheral we examined the cytolytic capability of these cells by using bronchial and vascular areas compared with naive mice an in vivo intratracheal cytotoxic T cell (CTL) killing assay (Figure 2a). To further examine the inflammatory and previously developed by us.45 Consistent with robust CD8

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Figure 2 Innate immune activation and minimal lung inflammation by intranasal inoculation of AdCh68Ag85A. Mice were inoculated via intranasal route with 1  107 plaque-forming units (PFUs) AdCh68Ag85A or AdHu5Ag85A or left unimmunized (naive). Lungs were harvested at 24 or 72 h after inoculation and processed for histopathologic examination. (a) Light hematoxylin and eosin (H&E) micrographs of original magnification  5 and  20 of lung sections are shown, representative of three mice per group per time point. (b) In separate experiments, the lungs were subject to bronchoalveolar lavage (BAL) and absolute numbers of alveolar macrophages (AM^s) and polymorphonuclear neutrophils (PMNs) in BAL were analyzed. The concentrations of (c) tumor necrosis factor-a (TNFa) and (d) interferon-b (IFNb) in BAL were determined by enzyme linked immunosorbent assay (ELISA). Data in b, c, d are expressed as the mean±s.e.m. of three to four mice per group per time point.

T-cell responses within the airway lumen by AdCh68Ag85A to a single peptide pool (p1-57) or 6 individual peptide pools vaccination (Figure 3b,c), 440% of CTL target killing was (p1-10, p11-20, p21-30, p31-40, p41-50, and p51-57) of Ag85A observed in the BAL at 2 weeks and the level of CTL declined by (Figure 4a). Consistent with the data in Figure 3, although both 4 weeks following vaccination (Figure 3d). In comparison, the AdCh68Ag85A and AdHu5Ag85A triggered high frequencies level of CTL activity by intranasal AdHu5Ag85A was signi- of antigen-specific CD8 T cells in response to single p1-57 ficantly lower at 2 weeks, but not at 4 weeks, than that by peptide pool, the levels of such responses were higher by AdCh68Ag85A (Figure 3d). These data suggest that respira- AdCh68Ag85A (Figure 4a). Significant T-cell responses were tory mucosal vaccination with AdCh68Ag85A is capable of also seen with stimulation by individual peptide pools of p11- inducing robust T-cell responses on the respiratory mucosal 20, p21-30, and p41-50 following AdCh68Ag85A and surface and in the lung that are overall stronger than AdHu5Ag85A vaccination (the p11-20 pool contains the AdHu5Ag85A vaccination. well-characterized immunodominant H-2d Ag85A CD8 T-cell epitope MPVGGQSSF or peptide 14/Ep-1). However, Respiratory mucosal AdCh68Ag85A immunization AdCh68Ag85A triggered noticeably much greater T-cell res- activates CD8 T cells of broadened epitope specificity ponses to p21-30 stimulation than AdHu5Ag85A (Figure 4a). As the chimpanzee adenovirus represents a novel These data suggest the emergence of an additional immuno- utilized as a TB vaccine platform, we investigated whether dominant Ag85A CD8 T-cell epitope, resulting from AdCh68Ag85A vaccination would activate the T cells reactive respiratory mucosal AdCh68Ag85A vaccination. We have to the same immunodominant and subdominant epitopes also examined the effects of these two vaccines in H-2b C57BL/6 as AdHu5Ag85A vaccination. To this end, naive mice were mice. At 2 weeks following intranasal vaccination, lung vaccinated i.n. with AdCh68Ag85A or AdHu5Ag85A. At 2 mononuclear cells were analyzed by intracellular cytokine weeks, lung mononuclear cells were analyzed by intracellular staining for IFNg-producing CD8 and CD4 T cells in response cytokine staining for IFNg-producing CD8 T cells in response to 6 individual peptide pools of Ag85A described as above.

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Figure 3 Robust T-cell responses in the lung by respiratory mucosal AdCh68Ag85A immunization. (a) Experimental schema. Intranasal immunization with 1 Â 107 plaque-forming units (PFUs) AdCh68Ag85A or AdHu5Ag85A was carried out and animals were killed at weeks 2 and 4 after immunization. (b) Ag85A tetramer-specific CD8 T-cell responses in the airway lumen (bronchoalveolar lavage (BAL)) and the lung interstitium (Lung). Representative dotplots depicting frequencies of tetramer þ CD8 T cells in the BAL and lung at week 2 and week 4 are shown. Bar graphs show absolute numbers of tetramer þ CD8 T cells per BAL and per lung at week 2 and week 4 after immunization. (c) Ag85A-specific interferon-g (IFNg)-producing CD8 T-cell responses in the BAL and lung. Representative dotplots depicting frequencies of IFNg þ CD8 T cells in the BAL and lung at week 2 and week 4 are shown. Bar graphs show absolute numbers of IFNg þ CD8 T cells per BAL and per lung at week 2 and week 4 after immunization. (d) Cytotoxic T-cell (CTL) killing activity in the airway lumen. In vivo intratracheal CTL assay was performed at week 2 and week 4 after immunization. Bar graphs show percent in vivo killing of carboxyfluorescein succinimidyl ester (CFSE)-labeled CTL target cells. Data in b, c, d are expressed as the mean±s.e.m. of three to four mice per group per time point. Data in b, c are representative of three independent experiments. **Po0.01; ***Po0.001 compared with AdHu5Ag85A.

Consistent with previous findings,46 we found no major histo- AdHu5 or AdCh68 vaccination (data not shown). However, compatibility complex class I (MHC I)-restricted Ag85A- there were very small frequencies of MHC II-restricted CD4 specific CD8 T-cell responses in B6 hosts following either T-cell responses detected that were comparable between

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Figure 4 Activation of CD8 T cells of broadened epitope specificity by respiratory mucosal AdCh68Ag85A immunization. Mice were immunized via intranasal route with 1x107 plaque-forming units (PFUs) AdCh68Ag85A or AdHu5Ag85A. Lungs were harvested at week 2 after immunization and CD8 T-cell responses to Ag85A peptide pools or peptide were determined by interferon-g (IFNg) intracellular cytokine staining (ICS). (a) Bar graph shows frequencies of CD8 þ IFNg þ cells in the lung responding to seven peptide pools of Ag85A. (b) Bar graph shows frequencies of CD8 þ IFNg þ cells in the lung responding to newly designed nine peptide pools. These new peptide pools were designed in such a way that each peptide is present only in three unique pools, according to the Matrix shown in the table. The newly emerged immunodominant peptide 29 is highlighted. (c) Representative dotplots show frequencies of CD8 þ IFNg þ cells in the lung responding to a conventional immunodominant Ag85A peptide Ep-1 and to a newly emerged immunodominant Ag85A peptide Ep-2. Bar graph shows absolute numbers of CD8 þ IFNg þ cells in the lung responding to Ep-1 and Ep-2 peptides. Data in a, b, c are expressed as the mean±s.e.m. of three to four mice per group, representative of two independent experiments. *Po0.05; **Po0.01; ***Po0.001 compared with AdHu5Ag85A.

AdHu5 and AdCh68 (Supplementary Table S1 matrix47 (Figure 4b, Matrix). In so doing, each peptide is online). The level of such MHC II-restricted responses in present only in three unique pools. Significantly raised T-cell B6 mice was comparable to that detected in BALB/c mice responses against pools p1, p6, and p9 were identified only in (data not shown). These data together suggest that AdHu5 and AdCh68Ag85A-vaccinated, but not AdHu5Ag85A-vaccinated, AdCh68 vaccinations predominantly generate high levels of animals (Figure 4b). Thus, the likely new candidate epitope was MHC I-restricted CD8 T-cell responses in BALB/c, but not in peptide 29, a 15-mer peptide highlighted in Figure 4b, Matrix C57BL/6, hosts. table. To further verify it, by using an Immune Epitope To further identify the potential additional CD8 T-cell Database (http://tools.immuneepitope.org/) we identified the epitopes present in the p21-30 and p41-p50 pools in BALB/c highest probable CD8 T-cell epitope sequence in peptide 29 to mice, a new set of 9 peptide pools with the peptides p21-p30 and be VYAGAMSGL (Ep-2). Using its synthesized 9-mer peptide, p41-p50 were developed with each pool containing 2–9 we confirmed its immunodominance by demonstrating a peptides according to a previously described peptide-mapping robust CD8 T-cell response induced only by intranasal

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Figure 5 Potent protection against pulmonary tuberculosis (TB) by respiratory mucosal AdCh68Ag85A immunization in naive hosts. (a) Naive mice were left unimmunized (naive) or immunized subcutaneously with Bacillus Calmette–Gue´rin; BCG), intranasally with 1x107 plaque-forming units (PFUs) AdCh68Ag85A (AdCh68) or with AdHu5Ag85A (AdHu5). At 4 weeks after immunization, mice were challenged with Mycobacterium tuberculosis (M.tb) and the lungs were harvested for analysis 4 weeks after infection. (a) Bar graph shows the colony-forming units (CFUs) of M.tb in the lung. (b) Representative histopathologic images from hematoxylin and eosin (H&E)-stained lung sections of three mice per group are shown at original magnification  1.6 and  5. Data in a are expressed as the mean±s.e.m. of four to five mice per group, representative of two independent experiments. **Po0.01; ***Po0.001.

AdCh68Ag85A, but not AdHu5Ag85A, vaccination (Figure 4c). respiratory mucosal AdCh68 vaccination, there was markedly In comparison, the CD8 T cells activated by both AdCh68Ag85A reduced overall granulomatous inflammation and tissue injury and AdHu5Ag85A vaccination well responded to stimulation in the lung of AdCh68-vaccinated animals, contrasting wide- by the common immunodominant peptide MPVGGQSSF spread pronounced granulomatous lesions in the lung of (Ep-1) (Figure 4c), although as shown in Figure 3c, unvaccinated animals (Figure 5b). Of note, the lung of AdCh68 AdCh68Ag85A triggered higher levels of responses. These animals had less immunopathology than the lung of AdHu5- data indicate that compared with AdHu5Ag85A, mucosal vaccinated animals (Figure 5b). Thus, compared with AdHu5 AdCh68Ag85A vaccination is able to activate CD8 T cells of lungs, the sizes of granulomatous lesion were smaller and there broader epitope specificity in H-2d Balb/c hosts. was less bronchial injury in the lung of AdCh68-vaccinated animals. These data together suggest that compared with its Respiratory mucosal AdCh68Ag85A immunization AdHu5 counterpart, respiratory mucosal AdCh68Ag85A provides robust protection against pulmonary tuberculosis immunization provides overall further improved protective in naive hosts immunity against pulmonary TB in the lung of naive animals. Having demonstrated the potent T-cell immunogenicity of AdCh68Ag85A, we next assessed its protective efficacy against Different from its AdHu5 counterpart, respiratory mucosal pulmonary TB in naive animals. To this end, naive mice AdCh68Ag85A immunization is able to provide potent were vaccinated as above with AdCh68Ag85A (AdCh68) or antituberculosis immunity in hosts with preexisting anti- AdHu5Ag85A (AdHu5). As a comparison, some mice were AdHu5 immunity in the lung subcutaneously (s.c.) vaccinated with BCG (BCG) or were not AdCh vectors have recently emerged to be an attractive vaccinated at all (naive). At 4 weeks after immunization, mice alternative to AdHu5 counterparts for the development of were challenged via the airway with virulent M.tbH37Rv. human vaccines because of prevalence of anti-AdHu5 immu- Protective efficacy in the lung was evaluated at 4 weeks after nity in humans.22,24,33,35 Indeed, it has been widely accepted challenge. Indeed, respiratory mucosal AdCh68 vaccination that anti-AdHu5 antibodies do not crossreact with AdCh.22,24 markedly reduced the levels of M.tb infection in the lung However, it remains largely unclear whether anti-AdHu5 compared with unvaccinated animals (Figure 5a). The magni- T cells may negatively affect the potency of AdCh-based tude of enhanced protection by AdCh68 was comparable to that vaccine.30 Of importance, little is understood about whether by AdHu5 counterpart or by parenteral BCG vaccination AdCh vector-based vaccine would remain effective when (Figure 5a). delivered via the respiratory tract—the natural route of human As the relative amount of M.tb-associated lung immuno- AdHu5 infection—in the context of local anti-AdHu5 pathology is an even more sensitive and reliable indicator of immunity. This is an important issue to address as Ad-based protection in some other animal models than in murine models TB vaccines were developed primarily as a novel platform for of TB, we examined and compared lung immunopathology. respiratory mucosal delivery in humans. We began to address In keeping with significantly improved protection by this issue first by examining to which extent the preexisting

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anti-AdHu5 immunity would negatively affect the T cell- To investigate whether the greater T-cell responses seen in activating potency of parenteral (intramuscular) vaccination the lung of WtAd-exposed, AdCh68-vaccinated animals would with AdC68Ag85A or AdHu5Ag85A. Thus, naive mice were lead to better immune protection from pulmonary TB, groups first exposed to wild-type AdHu5 virus (WtAd) via the of mice were exposed i.n. to WtAd and subsequently vaccinated intranasal route (exposed). Such respiratory WtAd exposure via the respiratory mucosal route with AdCh68 or AdHu5 led to significant levels of anti-AdHu5 immunity as exemplified vaccine as above and then challenged via the airway with by markedly elevated total anti-AdHu5 IgG and IgA antibodies virulent M.tbH37Rv (Figure 6e). Relative levels of protective and raised levels of AdHu5-neutralizing antibody titers both efficacy in the lung were assessed at 4 weeks after M.tb infection. locally in the lung (BAL) and systemically in the peripheral Consistent with the protection data shown in Figure 5a, blood (serum) (Supplementary Figure S1). At 4 weeks after (WtAd) unexposed animals were similarly protected in the lung WtAd infection, mice were vaccinated intramuscularly by intranasal AdCh68 or AdHu5 vaccination (Figure 6f). Also, with AdCh68Ag85A (AdCh68) or AdHu5Ag85A (AdHu5) consistent with the immunogenicity data (Figure 6d), (Figure 6a). The control animals were vaccinated similarly preexisting anti-AdHu5 immunity had little negative effects without WtAd exposure (unexposed). Ag85A-specific T-cell on immune protection of AdCh68-vaccinated animals, whereas responses were examined in the BAL, lung, and spleen. As it significantly reduced protection, leading to markedly expected,6,11,12 intramuscular AdCh68 or AdHu5 vaccination increased M.tb infection in the lung of AdHu5-vaccinated of unexposed animals failed to elicit any airway luminal T cells animals (Figure 6f). As the above set-up did not directly (BAL), whereas WtAd-exposed animals had similarly increased address whether preexposure to WtAd on its own had an effect BAL T cells following intramuscular AdCh68 or AdHu5 on immune protection, in a separate experiment mice were vaccination (Figure 6b). On the other hand, compared with exposed to WtAd and at 4 weeks after exposure mice were left unexposed animals, significantly increased numbers of CD8 T either unvaccinated or vaccinated with AdHu5Ag85A or cells were observed in the lung of WtAd-exposed intramuscular AdCh68Ag85A. At 4 weeks after vaccination, mice were AdC68 animals (Figure 6b). On the contrary, significantly challenged with M.tb and bacterial burden in the lung was reduced numbers of CD8 T cells were observed in the lung of assayed. As expected, compared with WtAd-unexposed and WtAd-exposed intramuscular AdHu animals (Figure 6b). unvaccinated mice (naive/unexposed), WtAd exposure alone Similarly, reduced CD8 T cells were seen in the spleen of only without vaccination (naive/exposed) had little significant WtAd-exposed AdHu5-vaccinated animals, but not WtAd- enhancing effects on protection against M.tb infection exposed AdCh68 vaccinated animals (Figure 6b). These data (Figure 6g). Importantly, compared with naive/exposed together support and extend the previous findings that hosts, AdHu5Ag85A vaccination in the presence of anti- preexisting circulating anti-AdHu5 immunity dampens the AdHu5 immunity (AdHu5/exposed) failed to provide signi- potency of parenterally administered AdHu5 gene transfer ficantly enhanced protection, whereas AdCh68Ag85A vaccina- vectors but not those based on chimpanzee adenovirus.24,48 tion in the presence of anti-Ad5 immunity (AdCh68/exposed) After demonstrating that different from AdHu5Ag85A remained robustly protective (Figure 6g). The above data vaccination, intramuscular AdCh68Ag85A vaccination can together suggest that respiratory mucosal AdCh68Ag85A indeed bypass the preexisting anti-AdHu5 immunity to induce immunization remains capable of triggering potent protec- potent T-cell responses, we next investigated to which extent tive T-cell immunity against pulmonary TB in the animals with this could be reproduced upon respiratory mucosal AdCh68 preexisting anti-AdHu5 immunity in the lung. In contrast, the vaccination in WtAd-exposed animals. To this end, mice were potency of respiratory mucosal immunization with its human exposed to WtAd via the intranasal route and at 4 weeks after counterpart AdHu5Ag85A vaccine is markedly reduced by WtAd infection, they were vaccinated i.n. with AdCh68 or such local anti-AdHu5 immunity. AdHu5 vaccine (Figure 6c). The (WtAd) unexposed naive mice were vaccinated i.n. as controls. T-cell responses were Respiratory mucosal AdCh68Ag85A immunization boosts subsequently examined in the BAL and lung. Of interest, protective T-cell immunity against pulmonary tuberculosis compared with unexposed animals, preexisting anti-AdHu5 in parenteral BCG-primed hosts immunity in the respiratory mucosa appeared to result in Like a number of other candidate TB vaccines that are currently reduced airway luminal (BAL) T-cell responses following in the clinical development, AdCh68Ag85A was developed for either respiratory mucosal AdCh68 or AdHu5 vaccination boost vaccination of parenteral BCG-primed humans. Having (Figure 6d). However, preexisting anti-AdHu5 immunity had systematically investigated the immunogenicity and protective little negative effects on T cells in the lung interstitium of efficacy of respiratory mucosal AdCh68Ag85A vaccination AdCh68-vaccinated animals, whereas it significantly reduced described above, we next set out to examine its effectiveness in T-cell responses in the lung of AdHu5-vaccinated animals boosting protective immunity following parenteral BCG (Figure 6d). Overall, intranasal AdCh68Ag85A vaccination priming. We first examined its effect on T-cell responses. triggered much greater antigen-specific T-cell responses in the To this end, naive mice were primed s.c. with BCG. At 4 weeks entire lung (both airway luminal and lung interstitial after BCG priming, mice were boosted i.n. with AdCh68Ag85A compartments) than intranasal AdHu5Ag85A vaccination (BCG/AdCh68) (Figure 7a). Control mice received only BCG in respiratory WtAd-exposed animals. (BCG) or AdCh (AdCh) vaccination. At 4 weeks after boosting,

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Figure 6 Potent protective immunity against pulmonary tuberculosis (TB) by respiratory mucosal AdCh68Ag85A immunization in hosts with preexisting anti-AdHu5 immunity in the lung. (a) Experimental schema for (b). Mice were exposed intranasally to wild-type AdHu5 virus (WtAd). The control mice were not exposed to WtAd (unexposed). At week 4 after WtAd exposure, mice were immunized intramuscularly with 1 Â 107 plaque-forming units (PFUs) AdCh68Ag85A (AdCh68) or AdHu5Ag85A (AdHu5) vaccine. Bronchoalveolar lavage (BAL), lungs, and spleens were collected for analysis at week 2 after parenteral immunization. (b) Bar graphs show absolute numbers of Ag85A tetramer þ and CD8 þ IFN-g þ T cells in the airway lumen (BAL), lung interstitium (Lung), and spleen. IFNg, interferon-g.(c) Experimental schema for (d). Mice were exposed intranasally to WtAd. The control mice were not exposed to WtAd (unexposed). At week 4 after WtAd exposure, mice were immunized intranasally with AdCh68 or AdHu5 TB vaccine. BAL and lungs were collected for analysis at week 4 after respiratory mucosal immunization. (d) Bar graphs show absolute numbers of Ag85A tetramer þ and CD8 þ IFN-g þ T cells in BAL and lung. (e) Experimental schema for (f,g). Mice were set up as described in c except that at week 4 after respiratory mucosal immunization, animals were challenged with Mycobacterium tuberculosis (M.tb) and the levels of infection in the lung were assessed 4 weeks after M.tb challenge. (f) Bar graph shows the colony-forming units (CFUs) of M.tb in the lung. (g) Mice were set up as described in e except that a group of mice exposed to WtAd but not immunized was included. Bar graph shows the CFUs of M.tb in the lung. Data in b, d are expressed as the mean±s.e.m. from three to four mice per group, representative of three independent experiments. Data in f are the mean±s.e.m. from four to five mice per group. *Po0.05; **Po0.01; ***Po0.001; NS, not significant P40.05 compared with the unexposed control.

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Figure 7 Further enhanced protective immunity against pulmonary tuberculosis (TB) by respiratory mucosal AdCh68Ag85A boost immunization in parenteral Bacillus Calmette–Gue´rin (BCG)-primed hosts. (a) Experimental schema for (b–d). Mice were prime-immunized subcutaneously (s.c.) with BCG. At week 4, animals were boost-immunized via intranasal (i.n.) route with 5 Â 107 plaque-forming units (PFUs) AdCh68Ag85A (BCG/AdCh68). Control groups were immunized only with BCG (BCG) or AdCh68Ag85A (AdCh68). Bronchoalveolar lavage (BAL), lungs, and spleens were collected at week 4 after boost immunization. (b–d) Bar graphs show absolute numbers of CD4 þ IFN-g þ and CD8 þ IFN-g þ T cells in (b) BAL, (c) lung, and (d) spleen. The cells were stimulated either with crude mycobacterial antigens (BCG/culture filtrate (CF)) or an Ag85A CD4 or CD8 peptide (Ag85A peptide). (e) Experimental schema for (f, g). Mice were set up as in a including BCG s.c., AdCh68 i.n., and BCG (s.c.)/AdCh68 (i.n.) groups except that at week 4 after boost immunization, mice were challenged with Mycobacterium tuberculosis (M.tb) and the lungs and spleens were collected for analysis. Furthermore, an additional control group was set up, receiving i.n. an empty control AdCh68 vector (vector). (f, g) Bar graphs show the colony-forming units (CFUs) of M.tb in the (f) lung and (g) spleen. Data in b, c, d are expressed as the mean±s.e.m. from three to four mice per group, representative of two independent experiments. Data in f, g are the mean±s.e.m. from five mice per group, representative of two independent experiments. *Po0.05; **Po0.01; ***Po0.001.

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cells isolated from the airway lumen (BAL), lung interstitium mucosal vaccination represents a powerful way to install the (lung), and spleen were stimulated with crude mycobacterial respiratory mucosa with T-cell immunity before M.tb exposure antigens (BCG/culture filtrate) or an Ag85A peptide and for enhanced lung protection.6,11–14 The potency of respiratory analyzed for antigen-specific, IFNg-producing CD4 and CD8 mucosal vaccination is not only limited to intracellular T-cell responses. Consistent with our previous findings,8 as its advantage has also recently been recognized parenteral BCG priming alone did not elicit airway luminal for immunity against lung cancer.49 Indeed, TB vaccine CD4 or CD8 T-cell responses (Figure 7b), whereas it induced a research community has recently identified the development level of crude mycobacterial antigen (BCG/culture filtrate)- of safe and effective respiratory mucosal TB vaccination reactive CD4 T-cell, but not CD8 T-cell, responses in the lung strategies to be a new priority.20 Such renewed effort is timely as (Figure 7c) and spleen (Figure 7d). On the other hand, as parenteral boosting with a vaccinia virus (MVA)-based TB expected, intranasal AdCh68 vaccination alone primarily vaccine was unable to improve lung protection in BCG vaccines activated Ag85A-specific CD8 T cells in the BAL and lung as recently shown in a phase 2b efficacy trial, the first of its kind (Figure 7b,c). In comparison, intranasal AdCh68 boost to test a new TB vaccine in the world.19 vaccination of parenteral BCG-primed animals (BCG/ Given its natural tropism to the respiratory mucosa and AdCh68) markedly increased multi-mycobacterial antigen- potency, recombinant AdHu5-based vaccine platforms have specific CD4 T cells in the airway lumen (BAL), lung, and proven to be most effective and promising for respiratory spleen (Figure 7b–d). It also increased both multi- mucosal vaccination against pulmonary TB based on abundant mycobacterial antigen- and Ag85A-specific CD8 T cells in preclinical data.11,41,42,45,50–52 However, the high prevalence of the BAL and spleen (Figure 7b–d). Together, these results preexisting anti-AdHu5 immunity in human populations has indicate that respiratory mucosal AdCh68Ag85A boost become an increasingly recognized limitation and challenge to immunization can potently enhance CD4 and CD8 T-cell application of AdHu5-based vaccines in humans.22,24,25,28–30,32 responses in the lung in parenteral BCG-primed animals. We have recently reported that preexisting circulating anti- To investigate whether intranasal AdCh68Ag85A-boosted T AdHu5 antibodies had no major effect on T-cell activation by cell immunity in the lung of BCG-primed hosts could lead to intramuscularly administered AdHu5Ag85A vaccine in a improved immune protection against pulmonary TB, mice human study.27 Nevertheless, as all trial volunteers had varying were subcutaneous BCG primed and received an intranasal degrees of preexisting anti-AdHu5 antibodies,27 we cannot boost with AdCh68 (BCG s.c./AdC68 i.n.) as above and firmly conclude that responses to the vaccine were not subsequently challenged via the airway with virulent dampened. Furthermore, as wild-type AdHu5 virus is primarily M.tbH37Rv (Figure 7e). Control groups were treated either a human respiratory , the anti-AdHu5 immunity with BCG alone (BCG s.c.) or AdCh68Ag85A alone (AdCh68 concentrated in lung mucosa could have greater negative effects i.n.) or an empty AdCh68 virus alone (vector i.n.). Relative on respiratory-mucosally administered AdHu5-based vaccine. levels of protective efficacy in the lung and spleen were assessed On the other hand, the results from Step HIV vaccine trials have at 4 weeks after M.tb infection. As expected, the vector control implied a safety concern in using AdHu5-based vaccine in those animals that were inoculated with the empty AdCh68 virus HIV high-risk populations who have high levels of preexisting were not protected, having high levels of M.tb infection in the anti-AdHu5 immunity.32,53,54 Thus, despite the big strides lung (Figure 7f), similar to the levels seen in infected naive made in basic and preclinical development of new TB vaccines, animals (Figure 5a). Again, as shown in Figure 5a, sub- there exists an important gap in meeting with the conditions cutaneous BCG priming alone or intranasal AdCh68 and effective applications in humans. These considerations vaccination alone each significantly enhanced protection, have prompted recently accelerated pace in the development of causing 0.7–0.8log M.tb colony-forming unit (CFU) reduc- heterologous chimpanzee adenovirus-based vaccine tion (Figure 7f). However, correlating with its improved T-cell technology.22,24,28,33,35 immunity (Figure 7b–d), AdCh68 boost vaccination of BCG- In this study, by using a new technology40 we have developed primed animals further improved protection, leading to an the first chimpanzee adenovirus-vectored TB vaccine as a novel B1.4log M.tb CFU reduction in the lung (Figure 7f). Similarly, candidate for respiratory mucosal immunization in humans. intranasal AdCh68 boosting also resulted in the best levels of We find that the ability of a single respiratory mucosal improved protection in the spleen (3log M.tb CFU reduction) AdCh68Ag85A immunization to activate T cells, provide compared with vector, BCG, or AdCh68 alone (Figure 7g). mucosal protection, and reduce M.tb-associated immuno- The above data together establish that respiratory mucosal pathology in naive animals with or without parenteral BCG AdCh68Ag85A boost immunization markedly enhances anti- priming is comparable to or even better than AdHu5Ag85A TB T-cell immunity in parenteral BCG-primed hosts. counterpart. Of importance, we provide compelling evidence that contrary to AdHu5Ag85A, respiratory mucosal DISCUSSION AdCh68Ag85A-induced protective T-cell immunity is mini- Mounting evidence suggests that any effective TB vaccination mally affected by anti-AdHu5 immunity preexisting locally in strategies ought to be able to close the ‘‘immunological gap’’ the lung. Given the scarcity of candidate TB vaccines currently instigated by M.tb infection whereby the appearance of T-cell amenable to safe and effective respiratory mucosal vaccination immunity is much delayed in the lung.6,7,11,12 Respiratory in humans,20 our study holds important implications. The

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findings from our study suggest that AdCh68-based TB vaccine crossreact with AdCh viral vectors. However, it is noteworthy is superior to the AdHu5-based counterpart and should be that in the majority of previous studies, preexisting anti-AdHu5 further developed clinically as an ideal TB vaccine candidate for immunity was generated by systemic AdHu5 infection. As respiratory mucosal immunization in humans. Given the ample AdHu5 virus is a respiratory pathogen in humans and recent experience and infrastructure well established by us and others, evidence suggests a differential immune-regulatory effect by the its expeditious translation to human studies and applications is mucosal route of induction of anti-AdHu5 immunity,61,62 in expected. this study we generated preexisting anti-AdHu5 immunity by Another noticeable advantage associated with AdCh68Ag85A respiratory route of wild-type AdHu5 infection that generated vaccine is its broadened CD8 T-cell epitope specificity noted in anti-Ad5-neutralizing antibody titers in both the lung and our study. Thus, respiratory mucosal AdCh68Ag85A immu- circulation, similar to the levels seen in humans.62 Thus, this nization results in the emergence of a dominant CD8 T-cell method is not only clinically relevant but is also relevant to the epitope corresponding to Ag85A amino acids 146–154 (Ep-2) investigation of AdCh-based respiratory mucosal TB vaccina- in addition to its strong inducibility of CD8 T-cell responses to tion strategies. Recently emerging clinical evidence has also the epitope spanning amino acids 70–80 (Ep-1). In comparison, raised the question of whether anti-AdHu5 T cells may its AdHu5 counterpart induces a strong response only to Ep-1, crossreact with the antigens from AdCh infection,30,32,35,63 thus with Ep-2 being a subdominant CD8 T-cell epitope. A broadened potentially negatively affecting the protective immunity by spectrum of dominant T-cell epitopes including Ep-2 by AdCh-based immunization. Indeed, we find that preexisting AdCh68-based immunization is considered an additional benefit anti-AdHu5 immunity present in the lung does have a to human applications as the epitopes within Ag85A amino acids dampening effect on AdCh68-induced T-cell responses within 141–160 were previously found to be the most immuno- the airway lumen. However, this dampening effect was found to dominant and MHC promiscuous in M.tb-infected humans.55 be limited only to the airway luminal space as the antigen- We believe that such broadened T-cell epitope specificity specific T-cell responses within the lung interstitium were not by AdCh68-based immunization may have contributed to affected. In contrast, preexisting local anti-AdHu5 immunity improved protection over that by AdHu5Ag85A immunization, significantly reduced the T cells in the lung interstitium of particularly with regard to reduced lung immunopathology. At AdHu5-vaccinated hosts. Thus, despite somewhat dampened the present time, the mechanisms for such difference between T-cell responses in the airway lumen by local anti-AdHu5 AdCh and AdHu5 vectors still remain to be fully understood. immunity, the lungs of AdCh68Ag85A-vaccinated hosts Although both AdCh and AdHu5 utilize the same coxsackievirus retained much greater numbers of M.tb antigen-specific and adenovirus receptor for infection,24,56 and we have observed T cells than AdHu5Ag85A-vaccinated counterparts. This their proinflammatory properties to be comparable, it is possible underpins one of the most important findings in our study that other differentially regulated intracellular signaling path- that preexisting anti-AdHu5 immunity in the lung does not ways may play a role. Indeed, previous studies suggest that the negatively affect AdCh68Ag85A-induced immune protection nature of formulation or vaccine vector has a profound effect on against pulmonary TB, whereas it significantly reduces the immunodominance of T-cell epitopes and the immune AdHu5Ag85A-mediated protection. In this study we have protective potential of activated T cells.57,58 This could be also demonstrated a dramatic boosting effect by AdCh68Ag85A attributed to antigen processing and presentation by antigen- immunization in BCG-primed animals. Respiratory mucosal presenting cells to CD8 T cells regulated by genes expressed by AdCh68 immunization boosted both multi-mycobacterial the vector.59,60 Thus, the broaden epitope recognition following antigen-specific CD4 T cells and Ag85A-specific CD8 T cells AdCh vaccination could be explained in part by the differential that led to markedly further enhanced protection. inherent prosperities of the viral vectors having a role in antigen In summary, we have developed a novel chimpanzee processing and canonical epitope presentation. However, adenovirus-based TB vaccine for respiratory mucosal immu- whether the broadened epitope specificity of AdCh68Ag85A nization and evaluated its potency in animal models of human vaccine may also hold true in humans requires further pulmonary TB with or without preexisting anti-AdHu5 investigation. Of note, despite enhanced T-cell responses by immunity in the lung. We provide compelling evidence that AdCh immunization relative to AdHu5 immunization, the chimpanzee adenovirus-based TB vaccine is superior to enhanced bacterial control by the two is comparable in WtAd- AdHu5-based counterpart and thus may represent an ideal unexposed hosts. This suggests that further improved T-cell candidate for respiratory mucosal immunization in humans. responses may not always translate into further improved Our findings also hold important implications in developing mycobacterial control. Nonetheless, such further improved effective virus-based vaccines against other respiratory infec- T-cell responses by AdCh immunization did lead to improved tion diseases and cancers for human applications. protection with respect to M.tb-associated histopathology. In keeping with previously published studies,24,38 we find METHODS that in contrast to AdHu5 vaccine the potency of parenterally Animals for in vivo models of immunization and pulmonary (intramuscularly) administered AdCh vaccine is not negatively tuberculosis. Female BALB/c mice, 6 to 8 weeks old, were purchased affected at all by preexisting systemic anti-AdHu5 immunity. from Charles River Laboratories (Charles River, St Constant, Quebec, This finding supports that anti-AdHu5 antibodies do not Canada) and housed in a specific pathogen-free level B facility at

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McMaster University. All experiments were carried out in accordance Splenocytes were isolated as described previously.41,50 All isolated cells with the guidelines from the Animal Research and Ethics Board at were resuspended in complete RPMI-1640 medium (RPMI 1640 McMaster University. supplemented with 10% fetal bovine serum, 1% penicillin– streptomycin, and 1% L-glutamine). Bioengineering of human and chimpanzee adenovirus-based . The construction of a recombinant repli- Cell stimulation, tetramer staining, intracellular cytokine staining, cation-deficient human type 5 adenovirus expressing an immuno- and flow cytometry. Mononuclear cells from BAL, lungs, or spleens dominant M.tb antigen 85A (AdHu5Ag85A) has been previously were cultured in U-bottom 96-well plates at a concentration of 20 described.41,64 This vaccine has been extensively evaluated as a million cells per ml for lungs and spleens and 0.5 million cells per ml for parenteral or respiratory mucosal TB vaccine in a wide range of BAL. For intracellular cytokine staining,42,45,50 cells were cultured in preclinical models.41,42,45,50–52 It has also recently been evaluated in a the presence of Golgi plug (5 mg ml À 1 brefeldin A; BD Pharmingen, phase 1 human study.27 San Jose, CA) with or without stimulation for 5–6 h with an Ag85A- A replication-deficient chimpanzee type 68 adenovirus was specific CD8 T cell-specific peptide (MPVGGQSSF)-Ep-1, a newly constructed to express an immunodominant M.tb antigen 85A identified Ag85A-specific CD8 T-cell peptide (VYAGAMSGL)-Ep-2, (AdCh68Ag85A) by using a direct cloning technology.40 The genetic or Ag85A-specific CD4 peptide (LTSELPGWLQANRHVKPTGS) at a Ag85A cassette containing the murine cytomegalovirus promoter, concentration of 1 mg per well. In selected experiments, cells were tissue plasminogen activator peptide signal, and polyA sequences used stimulated with crude BCG and M.tb CF at a concentration of 1 mg per for constructing AdCh68Ag85A (Figure 1a) is identical to the one for well for 24 h with last 6 h in the presence of Golgi plug. After AdHu5Ag85A. Briefly, a pShuttle plasmid DNA containing the Ag85A incubation, cells were washed and blocked with CD16/CD32 in 0.5% cassette was digested with I-Ceu1 and PI-Sce1. The released insert was bovine serum albumin/phosphate-buffered saline for 15 min on ice subcloned by an in-gel ligation technique into the I-Ceu1 and PI-Sce1 and then stained with the appropriate fluorochrome-labeled site of the genomic DNA clone pAdC68 containing the entire AdCh68 monoclonal antibodies. Cells were then processed according to the genomic sequences except the E1 and E3 genes. AdCh68Ag85A was manufacturer’s instructions (BD Pharmingen). The monoclonal packaged and propagated in 293 cells and purified by cesium chloride antibodies used included CD8a-phycoerythrin-Cy7, CD4-allophy- gradient centrifugation in the same way as for AdHu5Ag85A. The cocyanin-Cy7, IFN-g-allophycocyanin, and CD3-CyChrome. For Ag85A protein production and secretion by AdCh68Ag85A-infected tetramer immunostaining, a tetramer for the immunodominant CD8 mammalian cells was verified and compared with that in T-cell peptide (MPVGGQSSF) of Ag85A bound to the BALB/c major AdHu5Ag85A-infected cells by western immunoblotting using a histocompatibility complex class I allele H-2Ld (NIH Tetramer Core, monoclonal antibody specific for Ag85A (Figure 1b).41 Atlanta, GA) was used. Cells were washed and blocked with CD16/ CD32 in 0.5% bovine serum albumin/phosphate-buffered saline for Generation of preexisting anti-AdHu5 immunity in animals by 15 min on ice and stained with the tetramer for 1 h in the dark at room respiratory infection with wild-type human serotype 5 adenovirus. 8 temperature. Cells were then washed and stained with surface In some experiments, naive mice were infected i.n. with WtAd (1 Â 10 antibodies. Immunostained cells were run on an LSR II flow cytometer plaque-forming units (PFUs)) to generate preexisting anti-AdHu5 (BD Biosciences, San Jose, CA) and 250,000 events per sample were immunity before TB immunization. collected and analyzed on FlowJo software (version 9; Tree Star, Respiratory mucosal or parenteral immunization in animals with Ashland, OR). AdCh68Ag85A, AdHu5Ag85A, or BCG vaccine. Respiratory mucosal route of immunization was carried out by i.n. delivery of T-cell epitope mapping for identification of a newly emerged T-cell AdHu5Ag85A or AdCh68Ag85A (1 Â 107 PFUs per mouse).42,45,50 epitope following AdCh68Ag85A immunization. To investigate the In selected experiments, mice were immunized parenterally (intra- repertoire of antigen-specific CD8 T cells generated by AdC68Ag85A muscular) with the same dose of vaccine. In some experiments a larger and AdHu5Ag85A immunization, lung mononuclear cells from 7 immunized mice were ex vivo stimulated with pools of 57 peptides of dose (5 Â 10 PFUs) of AdCh68Ag85A was used. For prime-boost 27 47 immunization regimen, BCG (Pasteur) (1 Â 105 CFUs per mouse) was Ag85A by using a previously described method. In the initial inoculated s.c.50 for priming and the animals were then boosted i.n. screening, lung cells were stimulated separately with six peptide pools with Ad-based vaccine. (p1-p10, p11-p20, p21-p30, p31-p40, p41-p50, and p51-p57; each peptide is 15 amino acids in length with 10 amino acids overlapping) or M. tuberculosis preparation and animal models of pulmonary single peptide pool containing 1-57 peptides. Each peptide in every tuberculosis. M.tb bacilli were grown in Middlebrook 7H9 broth pool was kept at a concentration of 1 mg per well. Based on initial supplemented with Middlebrook oleic acid–albumin–dextrose–cat- screening, two peptide pools (p21-p30 and p41-p50) containing alase enrichment, 0.002% glycerol, and 0.05% Tween-80 for 10–15 potential CD8 T-cell epitopes were selected to design 9 sets of new days, aliquoted, and stored in À 70 1C until use.41,42,45,50 Before each peptide pools containing 2–9 peptides/pool in such a way that each in vivo use, M.tb bacilli were washed with phosphate-buffered saline peptide is present only in 3 unique pools. These peptide pools were containing 0.05% Tween-80 twice and passed through a 27-gauge then used for ex vivo stimulation of lung mononuclear cells from needle 10 times to disperse clumps. Pulmonary infection with immunized mice. Based on the magnitude of T-cell reactivity, a 15- M.tbH37Rv strain was carried out as previously described.41,42,45,50 amino-acid peptide potentially containing an immunodominant CD8 The levels of mycobacterial infection in the lung and spleen were T-cell epitope was identified and the corresponding amino acid determined by plating serial dilutions of tissue homogenates in tri- sequence was analyzed using the Immune Epitope Database (http:// plicates onto Middlebrook 7H10 agar plates. Plates were incubated at tools.immuneepitope.org/) for determining the highest probable CD8 37 1C for 15–17 days before colony enumeration. T-cell epitope sequence. A novel Ag85A-specific CD8 T cell-specific epitope sequence (VYAGAMSGL) was identified and the corre- Bronchoalveolar lavage and lung and spleen mononuclear cell sponding peptide synthesized and used for ex vivo antigen stimulation isolation. Mice were killed by exsanguination. Airway luminal cells as described above. were collected through exhaustive BAL as previously described.42,45 Subsequently, lungs were perfused by injecting Hank’s buffer through In vivo intratracheal cytotoxic T-cell assay. The intratracheal in vivo the right ventricle in order to remove intravascular mononuclear cells. CD8 CTL assay was conducted as previously described.45 Briefly, Lungs were digested with collagenase type 1 (Sigma-Aldrich, St Louis, 2.5 Â 106 antigen-pulsed and 2.5 Â 106 unpulsed, CFSE (carboxy- MO) at 37 1C in an agitating incubator. A single-cell suspension was fluorescein succinimidyl ester)-labeled splenic CTL target cells were obtained by crushing the digested tissue through 40 mm basket filter. combined to a final 40 ml of phosphate-buffered saline and transferred

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intratracheally to the airway of mice vaccinated with Ad-based vaccine & 2015 Society for Mucosal Immunology for 2 or 4 weeks. The animals were killed at 5 h after target cell transfer. Total cells isolated by bronchoalveolar lavage were run on the FACScan (BD Pharmingen) for assessment of the percentage of CFSE- REFERENCES labeled target cells. The percent in vivo killing of CFSE-labeled target 1. Cooper, A.M. Cell-mediated immune responses in tuberculosis. Annu. Rev. cells was defined as the relative loss of such cells after in vivo incubation Immunol. 27, 393–422 (2009). and thus was taken as the measure of CTL. 2. Kaufmann, S.H. Tuberculosis vaccine development: strength lies in tenacity. Trends Immunol. 33, 373–379 (2012). Assessment of lung inflammation by histology, BAL cytology, and 3. McShane, H. et al. BCG: myths, realities, and the need for alternative innate cytokine responses. Lung inflammation following respiratory vaccine strategies. 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Safety and efficacy of MVA85A, a new tuberculosis indicated as *Po0.05, **Po0.01, and ***Po0.001. A two-tailed vaccine, in infants previously vaccinated with BCG: a randomised, Student’s t-test was performed for pair-wise comparisons. One-way placebo-controlled phase 2b trial. Lancet 381, 1021–1028 (2013). analysis of variance and subsequent Tukey’s post hoc test were carried 20. Aerosol Vaccines For Tuberculosis Workshop Summary Group: out for the comparison of multiple groups. All analyses were per- Developing Aerosol Vaccines for Mycobacterium tuberculosis. Vaccine, formed by using GraphPad Prism software (GraphPad Software, San 2015 (in press). Diego, CA). 21. Xing, Z. & Lichty, B.D. Use of recombinant virus-vectored tuberculosis vaccines for respiratory mucosal immunization. Tuberculosis (Edinb) 86, SUPPLEMENTARY MATERIAL is linked to the online version of the paper 211–217 (2006). at http://www.nature.com/mi 22. Rollier, C.S., Reyes-Sandoval, A., Cottingham, M.G., Ewer, K. & Hill, A.V. Viral vectors as vaccine platforms: deployment in sight. Curr. Opin. ACKNOWLEDGMENTS Immunol. 23, 377–382 (2011). This work was supported by funds from the Canadian Institutes of Health 23. Song, K. et al. Genetic immunization in the lung induces potent local and Research, the Natural Sciences and Engineering Research Council of systemic immune responses. Proc. Natl. Acad. Sci. USA 107, 22213– Canada, the Canadian Foundation for Innovation, and Ontario Govern- 22218 (2010). ment. We are grateful to the technical advice provided by Dr Xiangyang 24. Lasaro, M.O. & Ertl, H.C. New insights on adenovirus as vaccine vectors. Zhou. We also acknowledge the kind provision of purified M.tb antigens, Mol. Ther. 17, 1333–1339 (2009). anti-Ag85A monoclonal antibodies, and Ag85A MHC class I tetramers by 25. Majhen, D. et al. 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